Project 2 will follow up on observations made in the previous funding period and further pursue whether virion-associated accessory proteins of HIV and SIV (i.e., Vpr, Vpx and Vif) can be used to target foreign proteins (including deleterious enzymes) to the virus particles as an avenue of anti-HIV therapy. The goals are to improve promising fusion constructs, investigate their mechanism of action, evaluate their antiviral activity in stable cell lines and primary target cells, and test additional fusion partners. The investigators also propose to use these fusion proteins as tools to probe the function of other virion components. The specific aims of this Project are : 1) To continue to characterize bacterial staphylococcal nuclease (SN) as a candidate fusion partner, and determine whether SN containing fusion constructs can be generated that exert a reproducible antiviral effect. The investigators propose to determine the intravirion location of Vpr/x-SN fusion proteins; to design new fusion constructs which target SN closer to the viral nucleic acid; to increase the availability of Ca2+ within the virion by packaging SN with calmodulin; to engineer Vpr/x-SN fusion properties with minimal packaging sequences; and to use Vif as an alternative fusion partner to target SN to the viral core. 2) To characterize further Vpr- and Vpx-based fusions with dominant negative mutants of HIV proteins (protease (PR), reverse transcriptase (RT) and integrase (IN)) which have a documented antiviral activity. The investigators propose to screen additional Vpr-IN-M fusion constructs for antiviral activity; generate T-cell lines that stably express candidate fusion constructs for antiviral activity; generate T-cell lines that stably express candidate fusion constructs (Vpr-IN-M, Vpx-PR-M) and assess them for permissiveness/restrictiveness to HIV/SIV replication; and transduce primary target cells (PBMCs and macrophages) with the most promising fusion constructs for challenge experiments with biologically relevant HIV/SIV strains. The antiviral activity of the most promising fusion constructs will be compared to that of other transgenes currently pursued in gene therapy approaches (e.g., transdominant rev, intracellular antibodies, etc.). 3) To improve the potency of existing antiviral constructs by testing additional fusion partners. The investigators propose to test E. coli RNase HI; uracil-DNA-glycosylase *and enzymatically inactive mutants of UDG); and Tat ( and negative dominant mutants of Tat).